1. Field of the Invention
This invention relates to methods of synthesis of single-stranded DNA or like polynucleotides of desired nucleotide sequence. More particularly, the invention relates to methods of synthesis of long stretches of single-stranded DNA of increased length and/or molecular weight.
2. Background Art
Short lengths of single-stranded DNA are used in a number of techniques employed in molecular biology, for example as primers employed in DNA amplification using the polymerase or ligase chain reaction (PCR or LCR), as primers used for DNA sequencing, or as labeled probes used for sequence identification in samples of DNA. Such short lengths of single-stranded DNA are normally produced by attaching a base nucleotide to a solid support and then linking one nucleotide at a time to the growing chain to create a nucleotide polymer of predetermined sequence. This is generally carried out within a commercial nucleic acid synthesizer. However, the greater the length of the DNA strand produced in this way, the lower is the purity of the resulting product (i.e. errors in the desired sequence are more likely as the sequence length increases). Acceptably pure sequences up to 100 bases in length may be synthesized with some difficulty, but sequences of more than 80 bases are not normally produced in this way. Indeed, sequences of only 20-35 base pairs are most commonly produced using such techniques. For short primers required for PCR or DNA sequencing, this limitation on sequence length is not usually a problem. However, other techniques may require sequences of 100 bases or more, e.g. those designed to introduce restriction sites into DNA molecules, those used in a multi-loci gene sequencing technique described in U.S. Pat. No. 6,197,510 issued to T. Vinayagamoorthy on Mar. 6, 2001, or those involving the synthesis of genes or for use in making microcircuits by means of nanotechnologies. Additionally, there are applications where DNA molecules of increased molecular weight or ionic charge for a given sequence length would be of benefit, e.g. in the multi-loci gene sequencing technique mentioned above.
There is therefore a need for a method of reliably synthesizing nucleic acid molecules of greater length and/or increased molecular weight or ionic charge than those produced by conventional techniques, while maintaining acceptable purity and the ability to determine a desired nucleotide sequence.